Audrey Minden

The Pak4 Protein Kinase Plays a Key Role in Cell Survival and Tumorigenesis in Athymic Mice

Pak4 is a member of the B group of p21-activated (Pak) kinases, originally identified as an effector protein for Cdc42. Although Pak4 is expressed at low levels in most adult tissues, it is highly overexpressed in tumor cell lines. Here, we show that Pak4 is also overexpressed in primary tumors, including colon, esophageal, and mammary tumors. Overexpression of Pak4 also leads to tumor formation in athymic mice, whereas deletion of Pak4 inhibits tumorigenesis. Although a constitutively active Pak4 mutant was previously shown to promote oncogenic transformation in cultured cells, our results are the first to show that Pak4 also promotes tumorigenesis in experimental animals. Furthermore, these results show for the first time that not only constitutively active Pak4, but also wild-type Pak4, is transforming, when experimental animals are used. These results are highly significant because wild-type Pak4, rather than activated Pak4, is overexpressed in tumor cells. Our results suggest that overexpression or activation of Pak4 is a key step in oncogenic transformation, due to its ability to promote cell survival and subsequent uncontrolled proliferation. The finding that Pak4 is up-regulated in so many types of cancers indicates that Pak4 may play a vital role in a wide range of different types of cancer. This makes it an attractive candidate for drug therapy for different types of cancer. (Mol Cancer Res 2008;6(7):1215–24)

P21 activated kinases: structure, regulation, and functions.

The p21 activated kinases (Paks) are well known effector proteins for the Rho GTPases Cdc42 and Rac. The Paks contain 6 members, which fall into 2 families of proteins. The first family consists of Paks 1, 2, and 3, and the second consists of Paks 4, 5, and 6. While some of the Paks are ubiquitously expressed, others have more restrictive tissue specificity. All of them are found in the nervous system. Studies using cell culture, transgenic mice, and knockout mice, have revealed important roles for the Paks in cytoskeletal organization and in many aspects of cell growth and development. This review discusses the basic structures of the Paks, and their roles in cell growth, development, and in cancer.

Targeted disruption of the Pak5 and Pak6 genes in mice leads to deficits in learning and locomotion

PAK6 is a member of the group B family of PAK serine/threonine kinases, and is highly expressed in the brain. The group B PAKs, including PAK4, PAK5, and PAK6, were first identified as effector proteins for the Rho GTPase Cdc42. They have important roles in filopodia formation, the extension of neurons, and cell survival. Pak4 knockout mice die in utero, and the embryos have several abnormalities, including a defect in the development of motor neurons. In contrast, Pak5 knockout mice do not have any noticeable abnormalities. So far nothing is known about the biological function of Pak6. To address this, we have deleted the Pak6 gene in mice. Since Pak6 and Pak5 are both expressed in the brain, we also generated Pak5/Pak6 double knockout mice. These mice were viable and fertile, but had several locomotor and behavioral deficits. Our results indicate that Pak5 and Pak6 together are not required for viability, but are required for a normal level of locomotion and activity as well as for learning and memory. This is consistent with a role for the group B PAKs in the nervous system.

Essential role for the Pak4 protein kinase in extraembryonic tissue development and vessel formation

Pak4 is a member of the group B family of Pak serine/threonine kinases, originally identified as an effector protein for the Rho GTPase Cdc42. Pak4 knockout mice are embryonic lethal and do not survive past embryonic day 11.5. Previous work on Pak4 knockout mice has focused on studying the phenotype of the embryo. Abnormalities in the extraembryonic tissue, however, are common causes of early embryonic death in knockout mice. Extraembryonic tissue associated with the Pak4-null embryos was therefore examined. Abnormalities in both yolk sacs and placentas resulted when Pak4 was deleted. These included a lack of vasculature throughout the extraembryonic tissue, as well as an abnormally formed labyrinthine layer of the placenta. Interestingly, epiblast-specific deletion of Pak4 using a conditional knockout system, did not rescue the embryonic lethality. In fact, it did not even rescue the extraembryonic tissue defects. Our results suggest that the extraembryonic tissue abnormalities are secondary to defects that occur in response to epiblast abnormalities. More detailed analysis suggests that abnormalities in vasculature throughout the extraembryonic tissue and the epiblast may contribute to the death of the Pak4-null embryos.

Systems-wide analysis of K-Ras, Cdc42 and PAK4 signaling by quantitative phosphoproteomics

Although K-Ras, Cdc42, and PAK4 signaling are commonly deregulated in cancer, only a few studies have sought to comprehensively examine the spectrum of phosphorylation-mediated signaling downstream of each of these key signaling nodes. In this study, we completed a label-free quantitative analysis of oncogenic K-Ras, activated Cdc42, and PAK4-mediated phosphorylation signaling, and report relative quantitation of 2152 phosphorylated peptides on 1062 proteins. We define the overlap in phosphopeptides regulated by K-Ras, Cdc42, and PAK4, and find that perturbation of these signaling components affects phosphoproteins associated with microtubule depolymerization, cytoskeletal organization, and the cell cycle. These findings provide a resource for future studies to characterize novel targets of oncogenic K-Ras signaling and validate biomarkers of PAK4 inhibition.

p120-catenin is a binding partner and substrate for Group B Pak kinases.

Pak5 is a member of the Group B p21‐activated kinases, which are effectors of the Rho family GTPases Cdc42 and Rac. Pak5 has been shown to promote cytoskeletal reorganization, inducing filopodia formation and neurite outgrowth in neuroblastoma cells. In this study, we used affinity chromatography followed by SDS–PAGE and mass spectrometry to identify potential downstream effectors of Pak5. Using this approach, we isolated p120‐catenin (p120), a known regulator of cytoskeletal reorganization and Rho GTPases. Using co‐immunoprecipitation assays we found that p120 preferentially interacts with Pak5 among the Group B Paks. Results from immunofluorescence studies revealed that Pak5 and p120 co‐localize in cells. Both Pak5 and constitutively active Pak4, the founding member of the Group B Paks, directly phosphorylate p120 in vitro. The phosphorylation was shown by Western blot and immunofluorescence to take place specifically on serine 288. This study is the first report of an upstream serine/threonine kinase that phosphorylates p120. J. Cell. Biochem. 110: 1244–1254, 2010. Published 2010 Wiley‐Liss, Inc.

PAK4 is required for regulation of the cell‐cycle regulatory protein p21, and for control of cell‐cycle progression

The serine/threonine kinase PAK4 regulates cytoskeletal architecture, and controls cell proliferation and survival. In most adult tissues PAK4 is expressed at low levels, but overexpression of PAK4 is associated with uncontrolled proliferation, inappropriate cell survival, and oncogenic transformation. Here we have studied for the first time, the role for PAK4 in the cell cycle. We found that PAK4 levels peak dramatically but transiently in the early part of G1 phase. Deletion of Pak4 was also associated with an increase in p21 levels, and PAK4 was required for normal p21 degradation. In serum‐starved cells, the absence of PAK4 led to a reduction in the amount of cells in G1, and an increase in the amount of cells in G2/M phase. We propose that the transient increase in PAK4 levels at early G1 reduces p21 levels, thereby abrogating the activity of CDK4/CDK6 kinases, and allowing cells to proceed with the cell cycle in a precisely coordinated way. J. Cell. Biochem. 112: 1795–1806, 2011.

A key role for Pak4 in proliferation and differentiation of neural progenitor cells

The Pak4 serine/threonine kinase regulates cytoskeletal organization, and controls cell growth, proliferation, and survival. Deletion of Pak4 in mice results in embryonic lethality prior to embryonic day 11.5. Pak4 knockout embryos exhibit abnormalities in the nervous system, the heart, and other tissues. In this study a conditional deletion of Pak4 was generated in order to study the function of Pak4 in the development of the brain. Nervous system-specific conditional deletion of Pak4 was accomplished by crossing mice with a floxed allele of Pak4 with transgenic mice expressing Cre recombinase under the control of the nestin promoter. The conditional Pak4 knockout mice were born normally, but displayed growth retardation and died prematurely. The brains showed a dramatic decrease in proliferation of cortical and striatal neuronal progenitor cells. In vitro analyses revealed a reduced proliferation and self-renewing capacity of neural progenitor cells isolated from Pak4 knockout brains. The mice also exhibited cortical thinning, impaired neurogenesis and loss of neuroepithelial adherens junctions. By the time the mice died, by 4weeks after birth, severe hydrocephalus could also be seen. These results suggest that Pak4 plays a critical role in the regulation of neural progenitor cell proliferation and in establishing the foundation for development of the adult brain.

Functional deficits in PAK5, PAK6 and PAK5/PAK6 knockout mice.

The p21-activated kinases are effector proteins for Rho-family GTPases. PAK4, PAK5, and PAK6 are the group II PAKs associated with neurite outgrowth, filopodia formation, and cell survival. Pak4 knockout mice are embryonic lethal, while Pak5, Pak6, and Pak5/Pak6 double knockout mice are viable and fertile. Our previous work found that the double knockout mice exhibit locomotor changes and learning and memory deficits. We also found some differences with Pak5 and Pak6 single knockout mice and the present work further explores the potential differences of the Pak5 knockout and Pak6 knockout mice in comparison with wild type mice. The Pak6 knockout mice were found to weigh significantly more than the other genotypes. The double knockout mice were found to be less active than the other genotypes. The Pak5 knockout mice and the double knockout mice performed worse on the rotorod test. All the knockout genotypes were found to be less aggressive in the resident intruder paradigm. The double knockout mice were, once again, found to perform worse in the active avoidance assay. These results indicate, that although some behavioral differences are seen in the Pak5 and Pak6 single knockout mice, the double knockout mice exhibit the greatest changes in locomotion and learning and memory.

Role for p21-activated kinase PAK4 in development of the mammalian heart

The serine-threonine kinase PAK4 plays a pivotal role in cell proliferation, survival, and control of the cytoskeleton. Mice that lack Pak4 die in midgestation prior to embryonic day E11 from unidentified causes. Analysis of PAK4 protein levels demonstrated that it was highly expressed in the whole embryo and in the developing heart but became low in the hearts of adult mice. In this study we analyzed development of the heart in conventional and conditional Pak4 knockout mice and embryos. We found that in conventional Pak4 knockout mice cardiogenesis is strongly affected from early developmental stages and by E9.5, hearts of Pak4−/− embryos developed multiple profound deficits. Conditional deletion of Pak4 in the progenitors of the secondary heart field led to abnormal development of the outflow tract, in which the pulmonary artery had a smaller diameter, and the aortal wall was thinner than in wildtype mice. The conditional knockout mice also displayed the characteristic enlargement of the right ventricles and right atria. Pak4−/− embryos and cardiomyocytes in which PAK4 was depleted exhibited low levels of LIMK1, a protein that plays key roles in cytoskeletal organization. Knock down of PAK4 in cultured cardiomyocytes led to severely compromised sarcomeric structure and deficits in contraction. These results indicate that PAK4 functions, including control of actin dynamics, are necessary for normal development of the heart.